Yersinia pestis, the bacterium responsible for plague, is a Category A pathogen, a significant biowarfare agent. If weaponized, it can easily gain direct access to the respiratory system to cause rapid death by pneumonic plague. Pneumonic plague has fatality rates over 50%, is highly contagious, and if the bacteria are engineered to resist antibiotics, is essentially unbeatable. As a countermeasure to such a threat, we propose to develop a library of bacteriophages ("phages") specific for Y. pestis that can kill multiple strains of this bacterium in a highly efficient manner. AvidBiotics has exclusive access to technology to generate families of phage with highly diverse tails capable of binding to mutant forms of bacteria, such as Bordetella. In this application, we propose to develop a library of phage to bind to, infect and kill mutant Y. pestis bacteria, anticipating potential mutations engineered into the bacteria to change their sensitivities to antibiotics or surface receptors for phage. This technology is based on the discovery of a family of diversity-generating retroelements (DGRs) that function to vary DNA sequences and the proteins they encode. These DGRs can be engineered into phages to diversify specifically the amino acid sequence of the precise receptor-binding site of the phage tail fiber, thereby generating a library of phages with more than a trillion different possible tails targeting Y. pestis cell surface receptors. By exposing such a phage library to mutant or weaponized plague bacteria, phages capable of infecting, multiplying and killing the bacteria could be identified, selected, isolated, amplified, aerosolized and then deployed to protect against or treat infections by weaponized Y. pestis. The objective of this application is to demonstrate the ability to utilize the DGR-system to precisely and extensively diversify the phage tail fiber sequences as a means to create novel phage with broad Y. pestis tropism. These studies will provide the foundation for future studies to generate a suitable, formulated pool of highly diverse phages that will serve as a reliable, productive source of diagnostic and therapeutic agents effective against weaponized plague bacteria. We anticipate that a Phase II follow-on project would entail developing a process to GLP manufacture and formulate representative members of the diverse phage library targeting Y. pestis and conduct pre-clinical safety studies to prepare a rapid response counter- terrorism system to manage pneumonic plague in humans. [unreadable] [unreadable] [unreadable]